This research proposal concerns the determination of the molecular steps in the transport of amino acids into prokaryotic cells. We have used genetic and biochemical approaches to identify four components, livJ, livK, livH, and livG, of high affinity leucine transport in E. coli. We are now applying cloning and DNA-sequencing techniques to these studies. A plasmid containing the entire leucine transport regulon has been prepared and will be used to study the in vitro synthesis and in vivo expression of the leucine transport components. Additional plasmids are being prepared which code for each of the two periplasmic leucine-binding proteins, leucine-binding proteins, leucine-specific and LIV-binding proteins, which serve as receptor sites for leucine transport in gram-negative bacteria. These periplasmic proteins are secretory proteins and we are using an in vitro synthesis system to study the processing of the binding proteins to the mature forms. The processing enzyme will be isolated and purified. DNA sequencing techniques are being used to determine the DNA sequence of the transport genes and their regulatory regions. The DNA sequence of the N-terminal portion of the leucine-binding protein genes will give us the amino acid sequence of the signal peptides of the precursor forms. These techniques combined with two-dimensional gel electrophoresis will be used to identify and characterize the membrane-bound components, coded for by livH and livG. The factors involved in the regulation of leucine transport, such as rho, will be examined and the regulatory role of secondary structure of the leucine transport messenger determined. Other transport systems will be examined using cloning, in vitro synthesis, and DNA sequencing techniques.